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281 lec31 mol_tech3

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Molecular techs. PCR and DNA sequencing methods

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281 lec31 mol_tech3

  1. 1. Lecture 31: Molecular techniques III. PCR and DNA sequencing Readings (chapter 10) Course 281
  2. 2. Lessons for life
  3. 3. AIMS • Understand the process of PCR and the components needed for the reaction. • Understand why PCR is important in molecular biology. • Understand the importance of DNA sequencing. • Understand Sanger sequencing method as an example of DNA sequencing.
  4. 4. Polymerase chain reaction • Polymerase Chain Reaction (PCR) allows the amplification (copying) of small amounts of DNA millions of copies. • The method was developed by Kary Mullis (1983) and he was awarded the Nobel Prize for his invention.
  5. 5. Polymerase chain reaction • The process of PCR is similar to the process of DNA replication except it is done in tubes rather than living cells. • It is considered in many cases the first step before any genetic analysis. • Many methods and applications involve PCR.
  6. 6. DNA replication and PCR • DNA replication in the cells involves making an identical copy of the genome (DNA). • PCR uses the same procedure but to generate millions of copies of a small section of the genome in a tube!
  7. 7. Why PCR? PCR is used: 1. To amplify small quantities of DNA. 2. For DNA quantification. 3. For genetic profile analyses: • RFLP • Microsatellite • Mitochondrial DNA genotyping and sequencing. 4. For sequencing small section of the genome or the genome.
  8. 8. Components What do we need to replicate (copy) DNA? 1. DNA template. 2. Building block of DNA (dNTPs). 3. DNA copier (an enzyme). 4. 3’OH (primer).
  9. 9. Components: (1) DNA template • The DNA sample you collect from a crime scene or the one under investigation is the DNA template. G A C T A T T A G C G C A T C G C G G C T A A T T A G C A T C G A T C G 5’ 5’ 3’ 3’ C TT A C C TG G CA T A C T G T G 5’3’ G AA T G G AC C GT A T G A C A C 5’ 3’ Each strand serves as a template for copying. Remember complementary base-pairing!
  10. 10. Components: (2) dNTP (building blocks) Do you remember? DNA is made of nucleotides!
  11. 11. Deoxyribonucleoside triphosphate (dNTP) H Four dNTPs serve as the building blocks of DNA (dATP, dTTP, dGTP, dCTP) Remember Nucleotides! Components: (2) dNTP (building blocks)
  12. 12. Deoxyribonucleoside triphosphate (dNTP) Why triphosphate? For the energy required to for the phosphodiester bond + H H Components: (2) dNTP (building blocks)
  13. 13. Components: (3) DNA copier (polymerase) • DNA polymerase is the DNA copier in the cell. • Uses the dNTPs (DNA building blocks) to make a complementary strand to the template. • Uses the available 3’-OH of a previous nucleotide and 5’phsphate from dNTP to form a phosphodiester bond.
  14. 14. Components: (3) DNA copier (polymerase) • Each time DNA Pol finds the correct complementary dNTP and catalyzes the reaction linking the new nucleotide. Remember DNA Pol needs 3’-OH
  15. 15. Primers are short piece of polynucleotide G C T A T T A G C C TG G CA T A C T G T G 5’ 5’ OH-3’ 3’ In order for the DNA copying machine to work and add nucleotides, a 3’-OH needs to be available to form a phosphodiester bond! Components: (4) primer (3’ OH)
  16. 16. PCR Process • Three steps are involved in PCR: 1. DNA template denaturation: separation of the two strands of DNA. 2. Primers annealing: small oligonucleotide attaches to each separated strand providing the 3’OH for DNA polymerase. 3. DNA polymerization (extension): DNA polymerase extends the primers on both strands and adds nucleotides.
  17. 17. PCR Process 1. DNA denaturation 2. Primers annealing 3. DNA extension
  18. 18. PCR cycles 1. DNA denaturation 2. Primers annealing 3. DNA extension Temp(C) 94 C 55-65 C 72 C What happens if we repeat this cycle many times? 2 copies 4 copies 8 copies 16 copies 32 copies Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5
  19. 19. PCR cycles 21 22 23 24 25 Exponential growth in the number of copies generated. The number of copies you get at the end of your PCR will be 2#cycles (236 cycles = 68 billion copies) 2 copies 4 copies 8 copies 16 copies 32 copies Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5
  20. 20. PCR – how many copies? #DNAcopies # cycles Threshhold Not enough DNA template No chemicals left in the reaction tube
  21. 21. Problems! There were some difficulties with this system: 1.Three water-baths with three different temperature. 2.DNA polymerase denatures at 94 C.
  22. 22. Problems! DNA denature (94 C) Primer annealing (55-65 C) Extension (72 C) • The sample has to be transferred into multiple water baths to accommodate the needed temperature.
  23. 23. Problems! DNA denature (94 C) Primer annealing (55-65 C) Extension (72 C) Adding DNA Pol DNA Pol denatures • DNA polymerase needs to be added in every cycle because DNA polymerase denatures at high temperature.
  24. 24. Improvement 1 • Using Thermus aquaticus (Taq) polymerase. • Taq polymerase is heat stable and the cycles can take place without the polymerase being destroyed during the denaturation phase
  25. 25. Improvement 2 Replacing old machine (water baths) with a thermocycler
  26. 26. To consider • Length and GC content of your primer. • Compatibility of your forward and reverse primers. • Primer’s sequences do not complement each other (primer dimer). • Annealing temperature of both primers should be the same. • Length of the target DNA piece ( the longer the target the longer the extension time). • DNA polymerase, primers and other chemicals’ concentration should be precisely calculated.
  27. 27. What is DNA sequencing? It is reading the letters of the book. It is reading the exact nucleotide sequence of the genome.
  28. 28. Sequencing methods available now! 1. Maxam and Gilbert chemical degradation method (extinct). 2. Sanger sequencing (dideoxy or chain termination method). 3. Illumina sequencing. 4. SOLiD sequencing. 5. Pyrosequencing. 6. Ion Torrent method. 7. Single molecule sequencing.
  29. 29. Why DNA sequencing? • DNA sequencing can be considered the ultimate characterization of gene(s) or fragment(s) of DNA. • DNA Sequencing is used for: • Mapping genomes • Determining gene structure and thus function • Detecting polymorphism (single nucleotide polymorphism SNP) • Analyzing genetic variation • Predicting the possible product(s) of DNA fragments • Many purposes depending on the questions one is asking
  30. 30. Sanger sequencing (the great method) 1.Fredrick Sanger has developed a sequencing method and received a Noble prize for it. 2.Sanger sequencing method is also called Chain Termination Method and Dideoxy sequencing method.
  31. 31. Sanger sequencing (the great method) • Employs: • specific primers • dNTPs • ddNTPs • DNA polymerase • DNA template
  32. 32. DNA synthesis + H H DNA synthesis requires the availability of a 3’-OH and energy
  33. 33. DNA synthesis Difference in OH location in sugar and consequences
  34. 34. DNA synthesis The absence of OH group on the 3’ carbon of the sugar blocks further addition of nucleotides
  35. 35. Sanger sequencing procedure ddGTP ddCTP ddATP ddTTP DNA Template Polymerase Excess dNTPs Primer
  36. 36. 3’5’5’5’ G5’ G A5’ AG A5’ AG A T5’ AG TA TAG TA T5’ C C5’ AG CTA TAG CTA T T5’ C Sanger sequencing procedure AG TA TGAATTC3’ 5’ 5’ G 5’ G A 5’ AG A 5’ AG A T 5’ AG TA T AG TA T5’ C C5’ AG CTA T AG CTA T T5’ C
  37. 37. A T G C + 3’ 5’ • Analysis using high resolution polyacrylamide gel electrophoresis. • Fragments are detected using radioactive markers and autoradiography. A C T G G T C A A T C G A T C G T A Sanger sequencing procedure
  38. 38. Sanger sequencing - Gel
  39. 39. • Each dideoxy nucleotide is attached to a florescent marker. • At the end of each cycle, a laser beam can detect the florescent marker and thus record the position of the nucleotide. Sanger sequencing - Automated
  40. 40. Sanger sequencing - Automated
  41. 41. Chromatogram - Automated
  42. 42. To know Polymerase chain reaction PCR DNA template dNTPs ddNTPs DNA polymerase Taq polymerase primer 3’-OH denaturation annealing extension DNA copies 2 #cycle thermocycler Sanger sequencing Dideoxy sequencing Chain termination sequencing Polyacrylamide gel Automated sanger sequencing Capillary electrophoresis chromatogram
  43. 43. Expectations • You know PCR’s components and process. • You know the phases of PCR and what happens in each phase. • You know how important it is for molecular applications. • You know DNA sequencing using Sanger method.
  44. 44. For a smile

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